PROJECT SUMMARY It is not known how T cells sense and respond to the stress of solid tumors, or how such responses shape dysfunction in CD8 tumor infiltrating lymphocytes (TILs). The endoplasmic reticulum (ER) is an organelle primed to sense environmental stress and to respond through initiation of downstream signal cascades. PKR-like ER kinase (PERK) is one of three ER sensors that enacts the cell response to stress. We were the first group to show that T cells in tumors experience the PERK-directed stress response. Using unique T cell-specific PERK KO mice (OT-1-Lckcre-PERKf/f) we discovered that PERK severely restricts the ability of T cells to control tumor growth. We now reveal groundbreaking preliminary data that illustrate that in response to the acute stress of the tumor microenvironment PERK prohibits T cell anti-tumor metabolism and restricts protein synthesis in CD8 TILs. Moreover, we show that CD8 TILs experience chronic ER stress associated with the PERK terminal unfolded protein response (UPR) enzyme ER oxidoreductase 1 (ERO1a) that induces dysregulated protein homeostasis (proteostasis) and impairs tumor control. This proposal aims to expand our preliminary data and develop the abovementioned novel discoveries into immunotherapeutic strategies to treat sarcomas with global implications for solid tumor cancer patients. To accomplish the aims proposed here we have developed a unique tumor microenvironment stress assay and sought out new approaches to apply to CD8 TIL biology to measure molecular changes that occur in T cells under tumor stress. In Aim 1 we will use T cells from OT-1-Lckcre-PERKf/f mice in our tumor stress assay paired with metabolomics to show that PERK is the central regulator of CD8 TIL metabolism. We will also use T cells from sarcoma patients and CRISPR/Cas9 in vitro gene editing of PERK to measure how PERK affects metabolism of human T cells under tumor microenvironment stress. The results are expected to reframe and advance our current understanding of CD8 TIL metabolism. In Aim 2 we will use T cells from OT-1-Lckcre-PERKf/f mice in our tumor stress assay paired with ribosome sequencing to discover the identity of actively translating mRNAs that are impacted by the tumor microenvironment. This work will also test the new concept that alternative routes of translation are sustainable in the stress of tumors. The results are expected to create a radical new paradigm of remodeling translation to improve immunotherapy. For Aim 3 we have created unique ERO1a-/- mice to formally define how ERO1a undermines proteostasis in CD8 TILs using an in vivo sarcoma model and proteomics. Successful completion of the aims will revolutionize the field of cancer immunotherapy by generating a new paradigm for the cell stress response as the primary factor that dismantles CD8 TIL anti-tumor efficacy in solid tumors.